scholarly journals Ribonucleotide incorporation enables repair of chromosome breaks by nonhomologous end joining

Science ◽  
2018 ◽  
Vol 361 (6407) ◽  
pp. 1126-1129 ◽  
Author(s):  
John M. Pryor ◽  
Michael P. Conlin ◽  
Juan Carvajal-Garcia ◽  
Megan E. Luedeman ◽  
Adam J. Luthman ◽  
...  
Keyword(s):  
Molecular Biology ◽  
Genome Stability ◽  
Nonhomologous End Joining ◽  
Complementary Strand ◽  
End Joining ◽  
Nhej Pathway ◽  
Central Dogma ◽  
Processing Enzymes ◽  
Chromosome Breaks ◽  
The Cost

The nonhomologous end–joining (NHEJ) pathway preserves genome stability by ligating the ends of broken chromosomes together. It employs end-processing enzymes, including polymerases, to prepare ends for ligation. We show that two such polymerases incorporate primarily ribonucleotides during NHEJ—an exception to the central dogma of molecular biology—both during repair of chromosome breaks made by Cas9 and during V(D)J recombination. Moreover, additions of ribonucleotides but not deoxynucleotides effectively promote ligation. Repair kinetics suggest that ribonucleotide-dependent first-strand ligation is followed by complementary strand repair with deoxynucleotides, then by replacement of ribonucleotides embedded in the first strand with deoxynucleotides. Our results indicate that as much as 65% of cellular NHEJ products have transiently embedded ribonucleotides, which promote flexibility in repair at the cost of more fragile intermediates.

scholarly journals Akt: A Double-Edged Sword in Cell Proliferation and Genome Stability

2012 ◽  
Vol 2012 ◽  
pp. 1-15 ◽  
Author(s):  
Naihan Xu ◽  
Yuanzhi Lao ◽  
Yaou Zhang ◽  
David A. Gillespie
Keyword(s):  
Genome Stability ◽  
Strand Break ◽  
Nonhomologous End Joining ◽  
Dependent Manner ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Cell Behaviour ◽  
Cell Cycles ◽  
Strand Breaks ◽  
Recombination Repair

The Akt family of serine/threonine protein kinases are key regulators of multiple aspects of cell behaviour, including proliferation, survival, metabolism, and tumorigenesis. Growth-factor-activated Akt signalling promotes progression through normal, unperturbed cell cycles by acting on diverse downstream factors involved in controlling the G1/S and G2/M transitions. Remarkably, several recent studies have also implicated Akt in modulating DNA damage responses and genome stability. High Akt activity can suppress ATR/Chk1 signalling and homologous recombination repair (HRR) via direct phosphorylation of Chk1 or TopBP1 or, indirectly, by inhibiting recruitment of double-strand break (DSB) resection factors, such as RPA, Brca1, and Rad51, to sites of damage. Loss of checkpoint and/or HRR proficiency is therefore a potential cause of genomic instability in tumor cells with high Akt. Conversely, Akt is activated by DNA double-strand breaks (DSBs) in a DNA-PK- or ATM/ATR-dependent manner and in some circumstances can contribute to radioresistance by stimulating DNA repair by nonhomologous end joining (NHEJ). Akt therefore modifies both the response to and repair of genotoxic damage in complex ways that are likely to have important consequences for the therapy of tumors with deregulation of the PI3K-Akt-PTEN pathway.

Repair of DNA Double-Strand Breaks by the Nonhomologous End Joining Pathway

2021 ◽  
Vol 90 (1) ◽  
Author(s):  
Benjamin M. Stinson ◽  
Joseph J. Loparo
Keyword(s):  
Genome Stability ◽  
Double Strand Breaks ◽  
Nonhomologous End Joining ◽  
Annual Review ◽  
Publication Date ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Break Site ◽  
Dna Ends

DNA double-strand breaks pose a serious threat to genome stability. In vertebrates, these breaks are predominantly repaired by nonhomologous end joining (NHEJ), which pairs DNA ends in a multiprotein synaptic complex to promote their direct ligation. NHEJ is a highly versatile pathway that uses an array of processing enzymes to modify damaged DNA ends and enable their ligation. The mechanisms of end synapsis and end processing have important implications for genome stability. Rapid and stable synapsis is necessary to limit chromosome translocations that result from the mispairing of DNA ends. Furthermore, end processing must be tightly regulated to minimize mutations at the break site. Here, we review our current mechanistic understanding of vertebrate NHEJ, with a particular focus on end synapsis and processing. Expected final online publication date for the Annual Review of Biochemistry, Volume 90 is June 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

scholarly journals Defective nonhomologous end joining blocks B-cell development in FLT3/ITD mice

Blood ◽  
2011 ◽  
Vol 117 (11) ◽  
pp. 3131-3139 ◽  
Author(s):  
Li Li ◽  
Li Zhang ◽  
Jinshui Fan ◽  
Kathleen Greenberg ◽  
Stephen Desiderio ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
B Lymphocyte ◽  
Cell Development ◽  
B Cell Development ◽  
Nonhomologous End Joining ◽  
End Joining ◽  
Lower Efficiency ◽  
Vdj Recombination ◽  
Nhej Pathway

Abstract We have generated an FLT3/ITD knock-in mouse model in which mice with an FLT3/ITD mutation develop myeloproliferative disease (MPD) and a block in early B-lymphocyte development. To elucidate the role of FLT3/ITD signaling in B-cell development, we studied VDJ recombination in the pro-B cells of FLT3/ITD mice and discovered an increased frequency of DNA double strand breaks (DSBs) introduced by the VDJ recombinase. Early pro-B cells from FLT3/ITD mice were found to have a lower efficiency and decreased accuracy of DSB repair by nonhomologous end joining (NHEJ), which is required for rejoining DSBs during VDJ recombination. Reduced NHEJ repair probably results from reduced expression of Ku86, a key component of the classic DNA-PK-dependent NHEJ pathway. In compensation, early pro-B cells from FLT3/ITD cells mice show increased levels of the alternative, and highly error-prone, NHEJ pathway protein PARP1, explaining the increase in repair errors. These data suggest that, in early pro-B cells from FLT3/ITD mice, impairment of classic NHEJ decreases the ability of cells to complete postcleavage DSB ligation, resulting in failure to complete VDJ recombination and subsequent block of B-lymphocyte maturation. These findings might explain the poor prognosis of leukemia patients with constitutive activation of FLT3 signaling.

scholarly journals Essential role for polymerase specialization in cellular nonhomologous end joining

2015 ◽  
Vol 112 (33) ◽  
pp. E4537-E4545 ◽  
Author(s):  
John M. Pryor ◽  
Crystal A. Waters ◽  
Ana Aza ◽  
Kenjiro Asagoshi ◽  
Christina Strom ◽  
...  
Keyword(s):  
Dna Synthesis ◽  
Nonhomologous End Joining ◽  
End Joining ◽  
Cellular Resistance ◽  
Strand Breaks ◽  
Chromosome Breaks ◽  
Repair Efficiency ◽  
The Face ◽  
Resistance To Ionizing Radiation ◽  
Unpaired Base

Nonhomologous end joining (NHEJ) repairs chromosome breaks and must remain effective in the face of extensive diversity in broken end structures. We show here that this flexibility is often reliant on the ability to direct DNA synthesis across strand breaks, and that polymerase (Pol) μ and Pol λ are the only mammalian DNA polymerases that have this activity. By systematically varying substrate in cells, we show each polymerase is uniquely proficient in different contexts. The templating nucleotide is also selected differently, with Pol μ using the unpaired base adjacent to the downstream 5′ phosphate even when there are available template sites further upstream of this position; this makes Pol μ more flexible but also less accurate than Pol λ. Loss of either polymerase alone consequently has clear and distinguishable effects on the fidelity of repair, but end remodeling by cellular nucleases and the remaining polymerase helps mitigate the effects on overall repair efficiency. Accordingly, when cells are deficient in both polymerases there is synergistic impact on NHEJ efficiency, both in terms of repair of defined substrates and cellular resistance to ionizing radiation. Pol μ and Pol λ thus provide distinct solutions to a problem for DNA synthesis that is unique to this pathway and play a key role in conferring on NHEJ the flexibility required for accurate and efficient repair.

scholarly journals Non-homologous end joining minimizes errors by coordinating DNA processing with ligation

2019 ◽  
Author(s):  
Benjamin M. Stinson ◽  
Andrew T. Moreno ◽  
Johannes C. Walter ◽  
Joseph J. Loparo
Keyword(s):  
Single Molecule ◽  
Genome Stability ◽  
Short Range ◽  
Genome Instability ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Synaptic Complex ◽  
Processing Enzymes ◽  
Non Homologous End Joining ◽  
Dna Ends

Genome stability requires efficient and faithful repair of DNA double-strand breaks (DSBs). The predominant DSB repair pathway in human cells is non-homologous end-joining (NHEJ), which directly ligates DNA ends1–5. Broken DNA ends at DSBs are chemically diverse, and many are not compatible for direct ligation by the NHEJ-associated DNA Ligase IV (Lig4). To solve this problem, NHEJ end-processing enzymes including polymerases and nucleases modify ends until they are ligatable. How cells regulate end processing to minimize unnecessary genomic alterations6 during repair of pathological DSBs remains unknown. Using a biochemical system that recapitulates key features of cellular NHEJ, we previously demonstrated that DNA ends are initially tethered at a distance, followed by Lig4-mediated formation of a “short-range synaptic complex” in which DNA ends are closely aligned for ligation7. Here, we show that a wide variety of end-processing activities all depend on formation of the short-range complex. Moreover, using real-time single molecule imaging, we find that end processing occurs within the short-range complex. Confining end processing to the Lig4-dependent short-range synaptic complex promotes immediate ligation of compatible ends and ensures that incompatible ends are ligated as soon as they become compatible, thereby minimizing end processing. Our results elucidate how NHEJ exploits end processing to achieve versatility while minimizing errors that cause genome instability.

scholarly journals Homeostasis in the Central Dogma of Molecular Biology: the importance of mRNA instability

2019 ◽  
Author(s):  
José E. Pérez-Ortín ◽  
Vicente Tordera ◽  
Sebastián Chávez
Keyword(s):  
Molecular Biology ◽  
Model Organism ◽  
Model Organisms ◽  
Published Data ◽  
Central Dogma ◽  
E Coli ◽  
Response Capacity ◽  
Cultured Human Cells ◽  
The Cost

AbstractCell survival requires the control of biomolecule concentration, i.e. biomolecules should approach homeostasis. With information-carrying macromolecules, the particular concentration variation ranges depend on each type: DNA is not buffered, but mRNA and protein concentrations are homeostatically controlled, which leads to the ribostasis and proteostasis concepts. In recent years, we have studied the particular features of mRNA ribostasis and proteostasis in the model organismS. cerevisiae. Here we extend this study by comparing published data from three other model organisms:E. coli, S. pombeand cultured human cells. We describe how mRNA ribostasis is less strict than proteostasis. A constant ratio appears between the average decay and dilution rates during cell growth for mRNA, but not for proteins. We postulate that this is due to a trade-off between the cost of synthesis and the response capacity. This compromise takes place at the transcription level, but is not possible at the translation level as the high stability of proteins,versusthat of mRNAs, precludes it. We hypothesize that the middle-place role of mRNA in theCentral Dogmaof Molecular Biology and its chemical instability make it more suitable than proteins for the fast changes needed for gene regulation.Graphical Abstract

scholarly journals Ku80 removal from DNA through double strand break–induced ubiquitylation

2008 ◽  
Vol 182 (3) ◽  
pp. 467-479 ◽  
Author(s):  
Lisa Postow ◽  
Cristina Ghenoiu ◽  
Eileen M. Woo ◽  
Andrew N. Krutchinsky ◽  
Brian T. Chait ◽  
...  
Keyword(s):  
Xenopus Laevis ◽  
Strand Break ◽  
Double Strand Break ◽  
Nonhomologous End Joining ◽  
Central Component ◽  
End Joining ◽  
Efficient Removal ◽  
Dsb Repair ◽  
Nhej Pathway ◽  
Egg Extract

The Ku70/Ku80 heterodimer, or Ku, is the central component of the nonhomologous end joining (NHEJ) pathway of double strand break (DSB) repair. Because Ku forms a ring through which the DSB threads, it likely becomes topologically attached to DNA during repair. The mechanism for its removal was unknown. Using a method to identify proteins recruited to DSBs in Xenopus laevis egg extract, we show that DSB-containing DNAs accumulate members of the Skp1–Cul1–F-box complex and K48-linked polyubiquitylated proteins in addition to known repair proteins. We demonstrate that Ku80 is degraded in response to DSBs in a ubiquitin-mediated manner. Strikingly, K48-linked polyubiquitylation, but not proteasomal degradation, is required for the efficient removal of Ku80 from DNA. This removal is DNA length dependent, as Ku80 is retained on duplex oligonucleotides. Finally, NHEJ completion and removal of Ku80 from DNA are independent from one another. We propose that DSB-induced ubiquitylation of Ku80 provides a mechanism to efficiently eliminate Ku from DNA for pre- and postrepair processes.

scholarly journals NHJ-1 Is Required for Canonical Nonhomologous End Joining in Caenorhabditis elegans

Genetics ◽  
2020 ◽  
Vol 215 (3) ◽  
pp. 635-651
Author(s):  
Aleksandar Vujin ◽  
Steven J. Jones ◽  
Monique Zetka
Keyword(s):  
Dna Damage ◽  
Caenorhabditis Elegans ◽  
Nonhomologous End Joining ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Dapi Staining ◽  
Nhej Pathway ◽  
C Elegans ◽  
Link Type ◽  
Recombination Pathway

DNA double-strand breaks (DSBs) are a particularly lethal form of DNA damage that must be repaired to restore genomic integrity. Canonical nonhomologous end joining (NHEJ), is a widely conserved pathway that detects and directly ligates the broken ends to repair the DSB. These events globally require the two proteins that form the Ku ring complex, Ku70 and Ku80, and the terminal ligase LIG4. While the NHEJ pathway in vertebrates is elaborated by more than a dozen factors of varying conservation and is similarly complex in other eukaryotes, the entire known NHEJ toolkit in Caenorhabditis elegans consists only of the core components CKU-70, CKU-80, and LIG-4. Here, we report the discovery of the first accessory NHEJ factor in C. elegans. Our analysis of the DNA damage response in young larvae revealed that the canonical wild-type N2 strain consisted of two lines that exhibited a differential phenotypic response to ionizing radiation (IR). Following the mapping of the causative locus to a candidate on chromosome V and clustered regularly interspaced short palindromic repeats-Cas9 mutagenesis, we show that disruption of the nhj-1 sequence induces IR sensitivity in the N2 line that previously exhibited IR resistance. Using genetic and cytological analyses, we demonstrate that nhj-1 functions in the NHEJ pathway to repair DSBs. Double mutants of nhj-1 and lig-4 or cku-80 do not exhibit additive IR sensitivity, and the post-IR somatic and fertility phenotypes of nhj-1 mimic those of the other NHEJ factors. Furthermore, in com-1 mutants that permit repair of meiotic DSBs by NHEJ instead of restricting their repair to the homologous recombination pathway, loss of nhj-1 mimics the consequences of loss of lig-4. Diakinesis-stage nuclei in nhj-1; com-1 and nhj-1; lig-4 mutant germlines exhibit increased numbers of DAPI-staining bodies, consistent with increased chromosome fragmentation in the absence of NHEJ-mediated meiotic DSB repair. Finally, we show that NHJ-1 and LIG-4 localize to somatic nuclei in larvae, but are excluded from the germline progenitor cells, consistent with NHEJ being the dominant DNA repair pathway in the soma. nhj-1 shares no sequence homology with other known eukaryotic NHEJ factors and is taxonomically restricted to the Rhabditid family, underscoring the evolutionary plasticity of even highly conserved pathways.

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